Circular site-directed mutagenesis

ABSTRACT

The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first, and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double-stranded circular DNA intermediates is transformed in suitable competent host cells and closed circular double-stranded DNA corresponding to the parental template molecules, but containing the desired mutation or mutations of interest, may be conveniently recovered from the transformed cells. The invention also provide kits for site-directed mutagenesis in accordance with methods of the present invention.

FIELD OF INVENTION

[0001] The invention is in the field of molecular biology, moreparticularly, in the field of the site-specific mutagenesis.

BACKGROUND

[0002] Site-directed mutagenesis has proved to a remarkably useful toolin molecular biology. Polynucleotides having pre-determined sequencesmay now be designed at will. Polymerase chain reaction (PCR) and variousother cyclic amplification reactions have been adapted for use insite-directed mutagenesis. Although site-directed mutagenesis throughPCR (the polymerase chain reaction) is widely used, PCR basedsite-directed mutagenesis techniques, have several shortcomings.

[0003] Several problems exist when trying to perform site-directedmutagenesis on double-stranded DNA molecules. These problems includestrand separation and selection against the parental (non-mutated) DNA.Efficient strand separation is important because in a typicalsite-directed procedure, a single polynucleotide primer containing thedesired sequence alteration must compete with the much longercomplementary strand for a hybridization site. Both physical andchemical methods for strand separation have been used. Physical methodsinclude the attachment of the DNA strands to a solid phase, such as aplastic bead (Hall, et al. Protein Eng. 4:601 (1991); Hultman, et al.Nucleic Acids Research 18:5107-5112 (1990); Weiner, et al. Gene126:35-41 (1993), or the use of heat as a denaturant (Landt, et al. Gene96:125-128 (1990); Sugimoto Analytical Biochemistry 179.:309-311 (1989).Chemical methods for strand separation usually rely on increasing the pHof the solution containing the DNA duplex (Weiner, et al. Gene 126:35-41(1993).

[0004] Following strand separation, the primer is annealed to theparental strand and used to initiate DNA replication. After replicationa means must be used to reduce the parental plasmid DNA contribution ofthe heteroduplex before or after cell transformation. Both in vivo andin vitro methods have been developed for this reduction. Innon-amplification based in vivo site-directed methods, the incorporationof dUTP into parental DNA during growth of the vector can be selectedagainst in dut*, ung* E coli cells (Kunkel Proc. Natl. Acad. Sci.(U.S.A.) 82:488-492 (1985). In vitro methods for selection of themutated strand include; i) unique restriction site elimination (Deng, etal. Analytical Biochemistry 200:81-88 (1992), ii), solid phasetechniques (where the parental DNA remains attached to the solid phase;Hultman, et al. Nucleic Acids Research 18:5107-5112 (1990); Weiner, etal. Gene 126:35-41 (1993), and iii) incorporation of modified bases inthe newly replicated DNA (Taylor et al. Nucleic Acids Research13:8765-8785 (1985); Vandeyar, et al. Gene 65:129-133 (1988).

[0005] When PCR has been used for site-specific mutagenesis, a strandseparation is accomplished during the high temperature denaturation stepin the cycling reaction. Selection against the parental DNA is usuallyaccomplished by decreasing the amount of starting template andincreasing the number of rounds of cycling. This increase in the numberof cycles has the adverse effect of increasing the rate of spontaneoussecond-site mutations, especially if an error-prone polymerase such asTaq DNA polymerase is used. In a typical experiment, the mutatedfragment is often subcloned from one vector to another. Often, differentantibiotic resistance markers are alternated or the mutated fragment isgel isolated. Descriptions of the use of the polymerase chain reaction(PCR) in site specific mutagenesis can be found in Hall, et al. ProteinEng. 4:601 (1991); Hemsley, et al. Nucleic Acids Research 17:6545-6551(1989); Ho, et al. Gene 77:51-59 (1989); Hultman, et al. Nucleic AcidsResearch 18:5107-5112 (1990); Jones, et al. Nature 344:793-794 (1990);Jones, et al. Biotechniques 12:528-533 (1992); Landt, et al. Gene96:125-128 (1990); Nassal, et al. Nucleic Acids Research 18:3077-3078(1990); Nelson, et al. Analytical Biochemistry 180:147-151 (1989);Vallette, et al. Nucleic Acids Research 17:723-733 (1989); Watkins, etal. Biotechniques 15:700-704 (1993); Weiner, et al. Gene 126:35-41(1993). Yao, et al. PCR Methods and Applications 1:205-207 (1992). Theuse of site-directed mutagenesis is also described in Weiner et al, Gene151:1/9-123(1994).

[0006] Given the many different methods of site-directed mutagenesisthat are in use, it is clear that no single technique currentlyavailable solves all of the problems associated with the site-directedmutagenesis. Given the state of the art, it is clearly of interest toprovide researchers (both industrial and academic) with useful newmethods of site-directed mutagenesis. To this end, the inventors havedeveloped new techniques for site-direct mutagenesis that havean-advantageous combination of features as compared to other techniquesfor site-directed mutagenesis. These useful features include: (1) lowsecondary mutation frequency, (2) high mutation efficiency, and (3) aminimal number of steps, thereby permitting the generation of host cellscontaining the mutant sequences in less than 24 hours.

SUMMARY OF INVENTION

[0007] The subject invention provides improved methods of site-directedmutagenesis involving linear cyclic amplification reactions. Theinvention provides extremely simple and effective methods of efficientlyintroducing specific mutations of interest into a target DNA.

[0008] The invention provides methods of introducing site-directedmutations into circular DNA of interest by means of mutagenic primerpairs that are selected so as to contain at least one mutation site withrespect to the target DNA sequence. The mutagenic primer pairs are alsoselected so as to be either completely complementary or partiallycomplementary to each other, wherein the mutation site (or sites) islocated within the region of complementarity of both mutagenic primers.

[0009] In the-methods of the invention, a mutagenic primer pair isannealed to opposite strands of a circular DNA molecule containing theDNA sequence to be mutagenized. After annealing, first and secondmutagenized DNA strands, each incorporating a member of the mutagenicprimer pair, are synthesized by a linear cyclic amplification reaction.The first and second mutagenized DNA strands synthesized are ofsufficient lengths for forming a double-stranded mutagenized circularDNA intermediate. The linear cyclic amplification reaction may berepeated for several cycles so as to generate a sufficient amount offirst and second mutagenized DNA strands for subsequent manipulations.After the linear cyclic amplification mediated synthesis step iscompleted, the reaction mixture is treated with a selection enzyme thatdigests the parental template strands, thereby enriching the reactionmixture with respect to the concentration of first and secondmutagenized DNA strands. The digestion step serves to digest parentalstrands that have annealed to the newly synthesized mutagenized DNAstrands and parental strands that have annealed to one another. Afterthe digestion step, the first and second mutagenized DNA strands arepermitted to hybridize to one another so as to form double-strandedcircular DNA intermediates. The double-stranded circular DNAintermediates are transformed into suitable competent host cells andclosed circular double-stranded DNA containing the desired mutation ormutations of interest may be conveniently recovered from the transformedcells.

[0010] The template digesting step in the methods of the invention maybe carried out in any of a variety of methods involving a selectionenzyme. The selection enzyme, e.g., a restriction endonuclease, is anenzyme that digests parental polynucleotides and does not digest newlysynthesized mutagenized polynucleotides. Either template polynucleotidesprior to replication are modified or polynucleotides synthesized duringreplication are modified so that the selection enzyme preferentiallycatalyzes the digestion of the parent template polynucleotide. In oneembodiment of the invention the polynucleotide for mutagenesis is dammethylated double-stranded DNA and the restriction enzyme used to digestparental polynucleotide strands is Dpn I.

[0011] Another aspect of the invention is to provide kits forsite-directed mutagenesis with high efficiency. The subject kits containreagents required for carrying the subject methods.

BRIEF DESCRIPTION OF THE DRAWING

[0012]FIG. 1. This figure provides a schematic diagram of an embodimentof the subject methods of site-directed mutagenesis. Step (A) shows acircular closed double-stranded plasmid. The “bull's eye” symbol is usedto indicate the target for mutagenesis. Step (B) shows the first andsecond mutagenic primer annealed to the circular closed double-strandedplasmid. The crosses indicate the mutagenic sites in the mutagenicprimers. The arrows indicate the direction of synthesis. Step (C) showsthe result of DNA synthesis from a linear cyclic amplification step. Thelighter shaded circular regions represent newly synthesized DNA that isadjoined to the mutagenic primers. The arrows indicate the direction ofsynthesis. Step (D) shows the mutagenized DNA strands that remain aftertreatment with a selection enzyme. The first and second mutagenizedstrands are shown as being annealed to form a double-strandedmutagenized circular DNA intermediate. Note the nicks on each strand.Step (E) shows the resultant mutagenized circular double-stranded DNAmolecules that are recovered after transforming competent cells with thedouble-stranded mutagenized circular DNA intermediate. Note that thecrosses in the diagram reflect the mutagenized sites that correspond tothe “bulls eyes” in Step (A).

[0013] Definitions

[0014] The term “linear cyclic amplification reaction,” as used herein,refers to a variety of enzyme mediated polynucleotide synthesisreactions that employ pairs of polynucleotide primers to linearlyamplify a given polynucleotide and proceeds through one or more cycles,each cycle resulting in polynucleotide replication. Linear cyclicamplification reactions used in the methods of the invention differsignificantly from the polymerase chain reaction (PCR). The polymerasechain reaction produces an amplification product that growsexponentially in amount with respect to the number of cycles. Linearcyclic amplification reactions differ from PCR because the amount ofamplification product produced in a linear cyclic amplification reactionis linear with respect to the number of cycles performed. Thisdifference in reaction prodcue accumulation rates is a result of usingmutagenic primers that are complementary or partially complementary toeach other. A linear cyclic amplification reaction cycle typicallycomprises the steps of denaturing the double-stranded template,annealing primers to the denatured template, and synthesizingpolynucleotides from the primers. The cycle may be repeated severaltimes so as to produce the desired amount of newly synthesizedpolynucleotide product. Although linear cyclic amplification reactionsdiffer significantly from PCR, guidance in performing the various stepsof linear cyclic amplification reactions can be obtained from reviewingliterature describing PCR including, PCR: A Practical Approach, M. J.McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methodsand Applications, by Innis, et al., Academic Press (1990), and PCRTechnology: Principals and Applications of DNA Amplification, H. A.Erlich, Stockton Press (1989). PCR is also described in many U.S.Patents, including U.S. Pat. Nos. 4,683,195, 4,683,202; 4,800,159;4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792, 5,023,171;5,091,310; and 5,066,584, which are hereby incorporated by referenceMany variations of amplification techniques are known to the person ofskill in the art of molecular biology. These variations include rapidamplification of DNA ends (RACE-PCR), amplification refectory mutationsystem (ARMS), PLCR (a combination of polymerase chain reaction andligase chain reaction), ligase chain reaction (LCR), self-sustainedsequence replication (SSR), Q-beta amplification, and stand displacementamplification (SDA), and the like. A person of ordinary skill in the artmay use these methods to modify the linear cyclic amplificationreactions used in the methods of the invention.

[0015] The term “mutagenic primer” refers to an oligonucleotide primerused in a linear cyclic amplification reaction, wherein the primer doesnot precisely match the target hybridization sequence. The mismatchednucleotides in the mutagenic primer are referred to as mutation siteswith respect to the mutagenic primer. Thus, during the amplificationreaction, the mismatched nucleotides of the primer are incorporated intothe amplification product thereby resulting in the synthesis of amutagenized DNA strand comprising the mutagenic primer that was used toprime synthesis mutagenizing the target sequence. The term“oligonucleotide” as used herein with respect to mutagenic primers isused broadly. Oligonucleotides include not only DNA but various analogsthereof. Such analogs may be base analogs and/or backbone analogs, e.g.,phosphorothioates, phosphonates, and the like. Techniques for thesynthesis of oligonucleotides, e.g., through phosphoramidite chemistry,are well known to the person ordinary skilled in the art and aredescribed, among other places, in Oligonucleotides and Analogues: APractical Approach, ed. Eckstein, IRL Press, Oxford (1992). Preferably,the oligonucleotide used in the methods of the invention are DNAmolecules.

[0016] The term “digestion” as used herein in reference to the enzymaticactivity of a selection enzyme is used broadly to refer both to (i)enzymes that catalyze the conversion of a polynucleotide intopolynucleotide precursor molecules and to (ii) enzymes capable ofcatalyzing the hydrolysis of at least one bond on polynucleotides so asto interfere adversely with the ability of a polynucleotide to replicate(autonomously or otherwise, or to interfere adversely with the abilityof a polynucleotide to be transformed into a host cell. Restrictionendonucleases are an example of an enzyme that can “digest” apolynucleotide. Typically, a restriction endonuclease that functions asa selection enzyme in a given situation will introduce multiplecleavages into the phosphodiester backbone of the template strands thatare digested. Other enzymes that can “digest” polynucleotides include,but are not limited to, exonucleases and glycosylases.

[0017] The term “selection enzyme” refers to an enzyme capable ofcatalyzing the digestion of a polynucleotide template for mutagenesis,but not significantly digesting newly synthesized mutagenizedpolynucleotide strands. Selection enzymes may differentiate betweentemplate and newly synthesized polynucleotides on the basis ofmodifications to either the parental template polynucleotide ormodifications to newly synthesized mutagenized polynucleotides.Selection enzymes suitable for use in the subject invention have theproperty of selectively digesting the parental strands of heteroduplexesformed between parental strands and the first or second mutagenized DNAstrands produced in the linear cyclic amplification reaction step.Examples of selection enzymes include restriction endonucleases andendoglycosylases.

[0018] The term “double-stranded mutagenized circular DNA intermediate”as used herein refers to double-stranded circular DNA structures formedby annealing he first mutagenized DNA strand formed in the subjectmethods to the second mutagenized DNA strand. When a double-strandedmutagenized circular DNA intermediate is transformed into a host cell,host cell enzymes are able to repair nicks (and possible small gaps) inthe molecule so as to provide a closed circular double-stranded DNA thatcorresponds to the original DNA molecule for mutagenesis that has beenmodified to contain the specific site-directed mutation or mutations ofinterest.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0019] The invention provides for, among other things, improved methodsfor site-directed mutagenesis. The improved methods of site-directedmutagenesis described herein provide for increased efficiency ofmutagenesis and the reduced introduction of secondary mutations. Themethods of the invention involve the use of pairs of complementary (orpartially complementary) mutagenic primers in linear cyclicamplification reactions. The methods of invention require a minimalnumber of DNA manipulations thereby decreasing the time and cost ofobtaining the desired mutants. In many instances, transformantscontaining DNA constructs with desired mutations may be obtained in asingle day (excluding the time to prepare the mutagenic primers).

[0020] The methods of the invention may be used to introduce one or moremutations in DNA sequences of interest. The DNA sequences of interestfor modification by the subject mutagenesis methods are necessarily partof a circular DNA molecule, i.e., the template molecule. The methods ofthe invention comprise the steps of annealing a first and secondmutagenic primer to the double-stranded circular molecule formutagenesis. The mutagenic primers are not generally phosphorylated, butmay be5′ phosphorylated. As the DNA molecule for mutagenesis isdouble-stranded, the annealing step is necessarily preceded by adenaturation step. The annealing step is typically part of a cycle of alinear cyclic amplification reaction. After annealing of the mutagenicprimers, first and second mutagenized DNA strands are synthesized fromthe first and second mutagenic primers, respectively. Synthesis of thefirst and second mutagenized DNA strands takes place during thesynthesis phase of a linear cyclic amplification reaction. The firstmutagenized DNA strand produced from the synthesis necessarily comprisesthe first mutagenic primer at its 5′ end. Similarly, the secondmutagenized DNA strand comprises the second mutagenic primer. The linearcyclic amplification reaction may be repeated through several cyclesuntil a sufficiency variety of first and second mutagenized DNA strandsare produced for the subsequent manipulations. After the linear cyclicamplification reaction steps, i.e., first and second. mutagenized DNAstrand synthesis are completed, the parental template DNA is digested byadding a selection enzyme. The selection enzyme serves to digestparental strand DNA. The parental strand DNA digested may be in the formof heteroduplexes formed between parental strands and the first orsecond mutagenized DNA strands produced in the linear cyclicamplification reaction step. Additionally, the parental strands digestedby the selection enzyme may consist of duplexes formed between parentalstrands. After the digestion step is completed, the first and secondmutagenized DNA strands are annealed to one another so as to produce adouble-stranded mutagenized circular DNA intermediate. Thedouble-stranded mutagenized circular DNA intermediates are subsequentlyused to transform a competent host cell. Transformed host cells may thenbe isolated as colonies and plasmids, i.e., closed circular DNAs,corresponding to the initial DNA molecules for mutagenesis, butcontaining the desired site-directed mutation or mutations, may beisolated from the transformed cells.

[0021] The previous paragraph has been primarily concerned with use ofdouble-stranded circular DNAs as targets for mutagenesis. A person ofordinary skill in the art may readily modify the procedure so as toprovide for site directed mutagenesis of circular single-stranded DNAs.In the case of a single-stranded circular DNA molecule for mutagenesis,only the first mutagenic primer is annealed in the initial step. Afterthe first primer is annealed synthesis of the first mutagenized strandproceeds so as to produce a double stranded circular DNA moleculecomprsing a first mutagenized DNA strand and the parentalsingle-stranded template. After the formation of the circular doublestranded molecule, the method may proceed as described in the previousparagraph.

[0022] The methods of the invention employ pairs of mutagenic primersconsisting of a first mutagenic primer and a second mutagenic primer.The mutagenic primers are about 20 to 50 bases in length, morepreferably about 25 to 45 bases in length. However, in certainembodiments of the invention, it may be necessary to use mutagenicprimers that are less than 20 bases or greater than 50 bases in lengthso as to obtain the mutagenesis result desired. The first and secondmutagenic primers may be of the same or different lengths; however, in apreferred embodiment of the invention the first and second mutagenicprimers are the same length.

[0023] The first and second mutagenic primers contain one or moremutagenic sites, i.e., mismatch locations with respect to the target DNAsequence to be mutagenized. The mutagenic site (or sites) may be used tointroduce a variety of mutation types into the DNA sequence formutagenesis. Such mutations include substitutions, insertions, anddeletions. The principle of site-directed mutagenesis with singleoligonucleotide primers is well known to the person of ordinary skill inthe art, and can be found, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring, Cold SpringHarbor, N.Y. (1989) and Wu et al., Recombinant DNA Methodology, AdademicPress, San Diego, Calif. (1989). This information may be used to designthe mutagenic sites in the first and second mutagenic primers employedin the subject methods.

[0024] The first and second mutagenic primers of the invention areeither completely complementary to each other or partially complementaryto each other. Preferably, the first and second mutagenic primers areselected so as to be completely complementary to each other. When thefirst and second mutagenic primers are partially complementary to eachother, the region of complementarity should be contiguous. Inembodiments of the invention in which the first and second mutagenicprimer are partially complementary to one another, the region ofcomplementarity must be sufficiently large to permit the mutagenicprimers to anneal to the DNA molecule for mutagenesis; preferably,although not necessarily, the region of complementarity is at least 50%of the length of the primer (50% of the larger primer when the first andsecond primer are of different lengths). The mutagenic sites of thefirst and second mutagenic primers are located in or near the middle ofthe primer. Preferably, the mutagenic sites are flanked by about 10-15bases of correct, i.e., non-mismatched, sequence so as to provide forthe annealing of the primer to the template DNA strands for mutagenesis.In preferred embodiments of subject methods, the GC content of mutagenicprimers is at least 40%, so as to increase the stability of the annealedprimers. Preferably, the first and second mutagenic primers are selectedso as to terminate in one or more G or C bases. The first and secondmutagenic primers for use in the subject invention are optionally 5′phosphorylated. 5′ phosphorylation may be achieved by a number ofmethods well known to a person of ordinary skill in the art, e.g., T-4polynucleotide kinase treatment. After phosphorylation, thephosphorylated primers must be purified prior to use in the methods ofthe invention so as to remove contaminants that may interfere with themutagenesis procedure. Preferred purification methods are fastpolynucleotide liquid chromatography (FPLC) or polyacrylamide gelelectrophoresis; however, other purification methods may be used. Thesepurification steps are unnecessary when non-phosphorylated mutagenicprimers are used in the subject methods.

[0025] First and second mutagenized DNA strands are synthesized by alinear cyclic amplification reaction. The exact parameter of eachportion of a cycle of the linear cyclic amplification reaction used mayvary in accordance with factors such as the DNA polymerase used, the GCcontent of the primers, DNA concentration, etc. Cycle parameters ofconcern include the time of each portion of the cycle (denaturation,annealing, synthesis) and temperature at which each portion of the cycletakes place. A person of ordinary skill in the art may obtain guidancein optimizing the parameters of the cyclic amplication reaction step forindividual experiments can be found in publications describing PCR. Thesynthesis phase of the linear cyclic amplification reactions used in thesubject mutagenesis methods should proceed for a length of timesufficient to produce first and second mutagenized DNA strandsequivalent in length (excluding insertions or deletions in the mutagenicprimers) to the circular DNA molecule for mutagenesis. When Pfu DNApolymerase is used to catalyze the linear cyclic amplification reaction,the synthesis phase of the linear cyclic amplification reactionoptimally occurs with a temperature range of 60°-68° C.; highertemperatures will result in the unwanted effect of mutagenic primerdisplacement.

[0026] The linear cyclic amplification reaction, i.e., the synthesisreaction, may be catalyzed by a thermostable or non-thermostablepolymerase enzyme. Polymerases for use in the linear cyclic amplifcationreactions of the subject methods have the property of not displacing themutagenic primers that are annealed to the template, thereby producing amutagenized DNA. strand of essentially the same length as the templatefrom which the newly synthesized strand was derived. Preferably, thepolymerase used is a thermostable polymerase. The polymerase used may beisolated from naturally occurring cells or may be produced byrecombinant DNA technology. The use of Pfu DNA polymerase (Stratagene),a DNA polymerase naturally produced by the thermophilic archaePyrococcus furiosus is particularly preferred for use in the linearcyclic amplification reaction steps of the claimed invention. Pfu DNApolymerase is exceptionally effective in producing first and secondmutagenized DNA strands of the appropriate length for formation of thedesired double-stranded mutagenized circular DNA intermediates. Examplesof other enzymes that may be used in linear cyclic amplificationinclude, but are not limited to, Taq polymerase, phage T7 polymerase,phage T4 polymerase, E. coli DNA polymerase I, Vent™ (New EnglandBiolabs, Beverly Mass.) DNA polymerase, Deep Vents DNA polymerase (NewEngland Biolabs, Beverly Mass.) Moloney Murine Leukemia Virus reversetranscriptase, and the like. When the DNA molecule for mutagenesis isrelatively long, it may be desirable to use a mixture of thermostableDNA polymerase, wherein one of the DNA polymerases has 5′-3′ exonucleaseactivity and the other DNA polymerase lacks 5′-3′ exonuclease activity.A description of how to amplify long regions of DNA using thesepolymerase mixtures can be found, among other places, in U.S. Pat. No.5,436,149, Cheng et al., Proc. Natl. Aca. Sci. USA 91:5695-9 (1994), andBarnes Proc. Natl. Aca. Sci. USA 91:2216-2220 (1994). In order todetermine whether or not a given polymerase (or multiple polymerasecomposition) is suitable for use in catalyzing the sythesis step of thelinear cyclic amplification reaction (under a given set of conditions),a simple assay using primers and circular template may be performed soas to determine if primer displacement occurs. Primer displacement mayreadily be detected by performing the gel electrophoresis anaylsis ofthe assay mixture.

[0027] Linear cyclic amplification reactions as employed in the methodsof the invention are preferably carried out with the minimum number ofamplification cycles required to produce the desired quantity of firstand second mutagenized DNA strands. Preferably the number of cycles inthe linear cyclic amplification reaction step is 30 cycles or less, morepreferably 20 or less cylces are performed, and even more preferably thenumber of cylces is between 10 and 20 (inclusive). However, thepreferred embodiment of cycles will vary in accordance with the numberof mutations sought to be introduced into the DNA molecule formutagenesis. Generally, the optimum number of reaction cycles willincrease with the complexity of mutations to be introduced into the DNAmolecule for mutagenesis. The use of a large number of amplificationcycles is troublesome because of the introduction of unwanted secondarymutations in the amplified sequences, i.e., mutations other than theintended site-directed mutagenesis target. Many polymerases used inlinear cyclic amplification reactions, especially Taq DNA polymerase,have relatively high error rates, thus increasing the number ofamplification cycles increases the number of secondary mutationsproduced. Prior to the invention, large numbers of amplification cycleswere required for linear cyclic amplification mutagenesis because of theneed to use a relatively low concentration of amplification target. Inthe past, low concentrations of amplification target were required sothat the amount of non-mutagenized product in a reaction mixture wassignificantly smaller than. the amount of desired mutagenized productproduced by linear cyclic amplification reactions, thereby reducing thenumber of transformants containing non-mutagenized polynucleotides. Thesubject methods of site-directed mutagenesis enable the use of acomparatively small number of amplification steps because relativelylarge amounts of template may be used without producing an unacceptablyhigh background of unmutagenized DNA molecules. The digestion stepserves to lower the background of unmutagenized DNA molecules. When alow, e.g., 5-10, number of amplification cycles are used in the linearcyclic amplification mutagenesis reaction, the amount of template DNAmolecule for mutagenesis should be increased so that a sufficient amountof mutagenized product is produced.

[0028] The methods of the subject invention comprise a “digesting” or“digestion” step in which the DNA molecules for mutagenesis, i.e., theparental template strands, are digested by a reaction catalyzed by anenzyme. This enzyme is referred to as a “selection enzyme.” In order toemploy a parental strand digestion step so as to reduce the parentalbackground in site-directed mutagenesis, a polynucleotide modificationstep must be employed prior to the parental strand digestion step. In apolynucleotide modification step for use in the subject methods ofsite-directed mutagenesis, either (1) one or more of the nucleotides ofthe parental template polynucleotides for mutagenesis are enzymatically(or chemically) modified and the first and second mutagenized DNAstrands synthesized during the replication reaction, e.g., the linearcyclic amplification reaction, are not modified or (2) one or more ofthe nucleotides of the first and second mutagenized DNA strandssynthesized during the linear cyclic amplification reaction areenzymatically (or chemically) modified and the nucleotides of theparental template DNA molecules for mutagenesis are not modified. Theprecise modification reaction step selected for use in a givenembodiment of the invention is selected in conjunction with the specificselection enzyme used in the digestion step so that the selection enzymecan digest the parental strand, i.e., the original templatepolynucleotides, and not significantly digest the newly synthesizedfirst and second mutagenized DNA strands.

[0029] The modifying step for use in conjunction with a parental stranddigestion step may comprise the process of exposing a DNA molecule formodification to a modifying agent. The modification step may be carriedout before the linear cyclic amplification reaction step or during thelinear cyclic amplification reaction step. The modifying agent may be amethylase enzyme that catalyzes the methylation of a base within thepolynucleotide of interest. Examples of suitable methylases for use inthe invention include dam methylase, dam methylase, Alu I methylase, andthe like. The modification reaction may take place in vivo or in vitro.In vivo methylation may be conveniently achieved by propagatingpolynucleotides in cells, either prokaryotic or eukaryotic, thatendogenously produce a suitable methylase enzyme. In a preferredembodiment of the invention, in vivo methylation is used to carry outthe modification step. The polynucleotide modification step may also beaccomplished by synthesizing polynucleotides with nucleotides comprisinga modified base, e.g., 6-methyl-ATP, rather than directly modifying apolynucleotide after the polynucleotide has been completely synthesized.When the modification reaction is a methylation reaction and theselection enzyme is a restriction endonuclease that requires methylatedbases for activity, the methylation step is preferably performed invivo. When the selection enzyme is a restriction endonuclease that doesnot cleave its recognition sequence when the recognition sequence of theenzyme is unmethylated, the modification reaction is preferably amethylation reaction performed in vitro by a polymerase catalyzing theincorporation of methylated nucleotides into a newly synthesizedpolynucleotide strand. When the selection enzyme used in the digestionstep is Dpn I, the modification step is preferably the methylation ofadenine to produce 6-methyl adenine (dam methylase) and the methylationreaction preferably takes place in vivo by propagating the DNA formutagenesis as a plasmid in a suitable prokaryotic host cell.

[0030] The digestion step involves the addition of a selection enzymethat is capable of digesting the parental, i.e., nonmutagenized, strandsof the DNA molecule for mutagenesis, but does not significantly digestnewly synthesized polynucleotides produced during a linear cyclicamplification mutagenesis. By performing the digestion step, the numberof transformants containing non-mutagenized polynucleotides issignificantly reduced. The parental strand digestion step involvesadding a selection enzyme to the reaction mixture after the linearcyclic amplification reaction has been completed. Selection enzymes maybe restriction endonucleases or other enzymes that are capable ofcatalyzing the digestion, e.g., cleavage, of parental strands in alinear cyclic amplification reaction, but do not significantly digestthe DNA strands newly synthesized during the linear cyclic amplificationreaction step. Restriction endonucleases for use in. the parental stranddigestion step are selected so as to be able to cleave the parentalstrands, but not significantly cleave newly synthesized polynucleotides.The restriction endonuclease selected for use in the digestion step may(1) require a specific modification of the parental strand that is notpresent on the first and second mutagenized DNA strands synthesizedduring the linear cyclic amplification mutagenesis reactions or (2) therestriction endonuclease selected for use in the parental stranddigestion step may be unable to digest polynucleotides that have beenmodified in a specific way and the first and second mutagenized DNAstrands synthesized during linear cyclic amplification reaction havesuch a modification (and the parental template polynucleotides, i.e, theDNA molecules for mutagenesis, lack the modification).

[0031] Restriction endonucleases are preferred for use as selectionenzymes in the digestion step. A preferred selection enzyme for use inthe parental strand digestion step is the restriction endonuclease DpnI, which cleaves the polynucleotide sequence GATC only when the adenineis methylated (6-methyl adenine). Other restriction endonucleasessuitable for use in the parental strand digestion step include Nan II,NmuD I, and NmuE I. However, restriction endonucleases for use asselection enzymes in the digestion step do not need to be isoschizomersof Dpn I.

[0032] In other embodiments of the invention, the selection enzymes usedin the digestion step are not restriction endonucleases. Other enzymesfor use as selection enzymes include uracil N-glycosylase. Uracildeglycosylase may be used as a selection enzyme by modifying a DNAmolecule for mutagenesis to contain one or more uracil bases rather thanthymidine. Uracil incorporation preferably occurs in vivo so that uracildeglycosylase may provide for the digestion of parental strands.Polynucleotides may be modified to as to contain thymidine residues by avariety of methods including DNA synthesis with dUTP as a DNA precursoror the replication of DNA in a dut⁻ ung⁻ strain of E. coli.Polynucleotides comprising uracil bases are sensitive todeglycosylation, i.e., digestion, by uracil N-glycosylase and otherenzymes with similar glycosylase activity. The use of uracilN-glycosylase is described, among other places in Kunkel, PNAS USA,82:488-492 (1985).

[0033] After the “digestion” step is completed or concurrent with the“digestion” step, i.e., the addition of the selection enzyme, the firstmutagenized DNA strands and the second mutagenized DNA strands areannealed to one another so as to produce a double-stranded mutagenizedcircular DNA intermediate. The formation of double-stranded mutagenizedcircular DNA intermediate takes place in accordance with conventionalprinciples of nucleic acid hybridization and may be performed under avariety of conditions. Conveniently, the annealing of the first andsecond mutagenized DNA strands so as to form a double-strandedmutagenized circular DNA intermediate may take place simultaneously withthe “digesting” step. The formation of the double-stranded circular DNAintermediates may take place in the same reaction vessel in which the“digesting” and/or the linear cyclic amplification reaction step takeplace. The process of forming double-stranded mutagenized circular DNAintermediates should proceed for a period of time sufficient to producea convenient number of double-stranded mutagenized circular DNAintermediates to provide a convenient number of clones in the subsequenttransformation steps. Generally, incubation for one to two hours at 37°C. will be sufficient in most embodiments of the invention. However,these time and temperature parameters may be readily varied by theperson or ordinary skill in the art so as to take into account factorssuch as DNA concentration, the GC content of the DNA molecules, etc.

[0034] After the double-stranded mutagenized circular DNA intermediateformation step is completed, the reaction mixture or a portion thereof,may be used to transform competent single-cell microorganism host cells.It is not necessary to perform a ligation reaction prior totransformation of the host cells. The absence of a ligation steprequirement serves to reduce the time and expense required to carry outthe methods of the invention as compared with conventional methods ofsite directed mutagenesis. The host cells may be prokaryotic oreukaryotic. Preferably the host cells are prokaryotic, more preferably,the host cells for transformation are E. coli cells. Techniques forpreparing and transforming competent single cell microorganisms are wellknow to the person of ordinary skill in the art and can be found, forexample, in Sambrook et al., Molecular Cloning: A Laboratory ManualColdspring Harbor Press, Coldspring Harbor, N.Y. (1989), HarwoodProtocols For Gene Analysis. Methods In Molecular Biology Vol. 31,Humana Press, Totowa, N.J. (1994), and the like. Frozen competent cellsmay be transformed so as to make the methods of the inventionparticularly convenient.

[0035] Another aspect of the invention is to provide kits for performingsite-directed mutagenesis methods of the invention. The kits of theinvention provide one or more of the enzymes or other reagents for usein performing the subject methods. Kits may contain reagents inpre-measured amounts so as to ensure both precision and accuracy whenperforming the subject methods. Kits may also contain instructions forperforming the methods of the invention. At a minimum, kits of theinvention comprise: a DNA polymerase (preferably Pfu DNA polymerase), aselection enzyme (preferably Dpn I), control primers, and controltemplates. Kits of the invention may contain the following items:individual nucleotide triphosphates, mixtures of nucleosidetriphosphates (including equimolar mixtures of DATP, dTTP, dCTP anddGTP), methylases (including Dam methylase), control linear cyclicamplification primers, bacterial strains for propagating methylatedplasmids (or phage), frozen competent cells, concentrated reactionbuffers, and the like. Preferred kits comprise a DNA polymerase,concentrated reaction buffer, a selection enzyme, a nucleosidetriphosphate mix of the four primary nucleoside triphosphates inequimolar amounts, frozen competent cells, control primers, and controltemplates. The terms “control template” and “control primer” as usedherein refer to circular double-stranded DNA molecules and mutagenicprimers, respectively that are selected to provide for easily detectablesite-directed mutagenesis by the methods of the invention. For example,a control template may comprise a lac Z gene with a point mutation andthe control primers may be designed to introduce a site-directedmutation that “repairs” the point mutation. As the lac Z phenotype iseasily detected on indicator media, e.g., X-gal, the efficiency of themutagenesis protocol may be easily monitored.

[0036] The invention having been described, the following examples areoffered by way of illustrating the invention and not by way oflimitation.

EXAMPLES

[0037] Control Reactions

[0038] A procedure for carrying out the site-directed mutagenesis ofplasmid pWhitescript™ 5.7-k.b. is given below. This procedure may bereadily adapted for the site-directed mutagenesis of other moleculesusing different primers. The plasmid pWhitescript™ 5.7-k.b. encodes amutant lacZ gene with point mutation that produce a lacZ minusphenotype. The primers are designed to “repair” this mutation so asproduce a plasmid that gives rise to a lacZ positive phenotype in E.coli grown on indicator medium. Accordingly, pWhitescript™ 5.7-k.b. maybe used as a control template in the kits of the invention

[0039] Setting Up the Reactions

[0040] 1. Synthesize two complementary oligonucleotides containing thedesired mutation, flanked by normal nucleotide sequence, i.e., first andsecond mutagenic primers. Optionally, the primers are 5′ phosphorylatedand gel purified prior to use in the following steps.

[0041] 2. Prepare the control reaction as indicated below:

[0042] 5 μl of 10× reaction buffer

[0043] 3 μl (3 ng, 0.001 nM) of pwhitescript™ 5.7-k.b.

[0044] control template (1 ng/μl)

[0045] 1.25 μl (125 ng, 22 nM) of oligonucleotide

[0046] control primer #1 [34-mer (100 ng/μl)]

[0047] 1.25 μl (125 ng, 22 nM) of oligonucleotide

[0048] control primer #2 [34-mer (100 ng/μl)]

[0049] 1 μl of 10 mM dNTP mix (2.5 mM each NTP)

[0050] Double-distilled water (ddH₂O) to a final volume of 50 μl .

[0051] Then add:

[0052] 1 μl of native Pfu DNA polymerase (2.5 U/μl)

[0053] 3. Prepare the sample reaction(s) as indicated below:

[0054] A series of sample reactions using various concentrations ofdsDNA template ranging from 2 to 8 ng (e.g., 2, 4, 6 and 8 ng of dsDNAtemplate) should be set up in order to determine the optimum amount.

[0055] 5 μl of 10× reaction buffer

[0056] 2-8 ng of dsDNA template

[0057] 125 ng of oligonucleotide primer #1

[0058] 125 ng of oligonucleotide primer #2

[0059] 1 μl of 10 mM dNTP mix (2.5 mM each NTP)

[0060] ddH₂O to a final volume of 50 μl

[0061] Then add:

[0062] 1 μl of native Pfu DNA polymerase (2.5 U/μl)

[0063] 3. Overlay each reaction with 30 μl of mineral oil. TABLE ICircular Site-Directed Mutagenesis Cycling Parameters Segment CyclesTemperature Time 1 1   95° C. 30 Seconds 2 10-16 95° C. 30 Seconds 50°C.  1 minute 68° C.  2 minutes/kb of plasmid length

[0064] Cycling the Reactions and Digesting the Products

[0065] 1. Thermal cycle each reaction using the cycling parameters areoutlined in Table I.

[0066] 2. Repeat segment 2 of the cycling parameters 10-16 times,depending on the type of mutation desired (i.e., 10 cycles for pointmutations, 12 cycles for single amino acid changes and 16 cycles formultiple amino acid deletions or insertions).

[0067] 3. Following linear amplification, place the reaction on ice for2 minutes to cool the reaction to ≦37° C.

[0068] Note In the following digestion step, it is important to insertthe pipet tip below the mineral oil overlay when adding the Dpn Irestriction enzyme to the reaction tubes.

[0069] 4. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly toeach amplification reaction below the mineral oil overlay with a pipettip.

[0070] 5. Gently and thoroughly mix each reaction mixture by pipettingthe solution up and down several times. Spin down the reaction mixturesin a microcentrifuge for 1 minute and immediately incubate each reactionat 37° C. for 1-2 hours to digest the parental (i.e., the nonmutated)supercoiled dsDNA.

[0071] Transforming into Epicurian Coli XL2-Blue Ultracompetent™ Cells(available from Stratagene)

[0072] The following protocol has been used successfully fortransforming E. coli with pBluescript®-derived plasmids encodingampicillin or chloramphenicol resistance. Transformation ofkanamycin-resistance-encoding plasmids require a 30- to 45-minuteoutgrowth after 10-fold dilution of the ultracompetent cells with SOCmedium (see Media and Reagent Preparation) between steps 3 and 4 of thetransformation protocol described in Sambrook et. al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989). Other selections may require a numberof similar outgrowth periods.

[0073] 1. Gently thaw the Epicurian Coli XL2-Blue™ ultracompetent cellson ice. For each control and sample reaction to be transformed, aliquotapproximately 50 μl of the ultracompetent cells to a prechilled Falcon®2059 polypropylene tube.

[0074] 2. Add 1 μl of the Dpn I-treated DNA from each control and samplereaction to separate aliquots of the ultracompetent cells and swirlgently to mix. Incubate the transformation reactions on ice for 30minutes, swirling periodically throughout the incubation.

[0075] As an optional step, verify the transformation efficiency of theEpicurian Coli XL2-Blue ultracompetent cells by adding 1 Al of the pUC18control plasmid (0.1 ng/μl) to a 50 μl aliquot of the ultracompetentcells and incubating as indicated above.

[0076] 3. Heat pulse the transformation reactions for 45 seconds at 45seconds at 42° C. and then place the reactions on ice for 2 minutes.This heat pulse has been optimized for the Falcon 2059 polypropylenetubes.

[0077] 4. Immediately plate the transformation reactions as outlinedbelow:

[0078] a. Plate the entire volume of the control transformation reactionand only 5 μl of the pUC18 control transformation reaction (ifperformed) on LB-ampicillin-methicillin agar plates (see Media andReagent section below) that have been spread with 20 μl of 10% (w/v)X-gal and 20 μl of 100 mM IPTG.

[0079] Note: Do not mix IPTG and X-gal, since these chemicals willprecipitate. X-gal should be prepared in dimethylformamide (DMF) and theIPTG should be prepared in filter-sterilized dH20.

[0080] b. Plate the entire volume of each sample transformation reactionon agar plates containing the appropriate antibiotic that is conferredby the plasmid vector being transformed.

[0081] 5. Incubate the transformation plates at 37° C. for >16 hours.

[0082] The expected colony number should be at least 50 colonies.Greater than 80% of the mutagenized control colonies should contain themutation and appear as blue colonies on agar plates containing IPTG andX-gal.

[0083] The mutagenesis efficiency (ME) for the pWhitescript 5.7-kbcontrol template is calculated by the following formula:${ME} = {\frac{{Number}\quad {of}\quad {blue}\quad {colony}\quad {forming}\quad {units}\quad ({cfu})}{{Total}\quad {number}\quad {of}\quad {colony}\quad {forming}\quad {units}\quad ({cfu})} \times 100\%}$

MEDIA AND REAGENTS TE Buffer 10X Reaction Buffer 10 mM Tris-HCl (pH 7.5)100 mM KCl  1 mM EDTA  60 mM (NH₄) 2SO₄ 200 mM Tris-HCl (pH 8.0)  20 mMMgCl₂ 1% Triton ® X-100 100 μg/ml nuclease-free bovine serum albumin(BSA) LB Agar (per Liter) LB-Ampicillin-Methicillin Agar 10 g of NaCl(per Liter) 10 g of tryptone (use for reduced  5 g of yeast extractsatellite colony 20 g of agar formation) Add deionized H₂O to a 1 literof LB agar final volume of 1 liter Autoclave Adjust pH to 7.0 with 5 Ncool to 55° C. NaOH Add 20 mg of filter-sterilized Autoclave ampicillinPour into petri dishes Add 80 mg of filter-sterilized (−25 ml/100-mmplate) methicillin Pour into petri dishes (−25 ml/100-mm plate) SOBMedium (per Liter) SOC Medium (per 100 ml) 20.0 g of tryptone SOB medium 5.0 g of yeast extract Add 1 ml of a 2 M filter-  0.5 g of NaClsterilized glucose Autoclave solution or 2 ml of 20% Add 10 ml of 1 MMgCl₂ (w/v) glucose prior to and 10 ml of 1 M use MgS)₄/liter of SOBmedium Filter sterilize prior to use Filter sterilize

INCORPORATION BY REFERENCE

[0084] All patents, patents applications, and publications cited areincorporated herein by reference.

[0085] Equivalents

[0086] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.Indeed, various modifications of the above-described makes for carryingout the invention which are obvious to those skilled in the field ofmolecular biology or related fields are intended to be within the scopeof the following claims.

What is claimed is:
 1. A method of introducing a specific mutation intoa selected DNA molecule for mutagenesis, wherein said DNA molecule is adouble-stranded circular DNA molecule, said method comprising the stepsof: annealing a first mutagenic primer and a second mutagenic primer tosaid DNA molecule, wherein said first mutagenic primer comprises aregion that is complementary to the second mutagenic primer and whereinsaid first and second mutagenic primers, synthesizing by means of alinear cyclic amplification reaction a first mutagenized DNA strandcomprising said first mutagenic primer, and a second mutagenized DNAstrand comprising said second mutagenic primer, wherein the firstmutagenized DNA strand and the second mutagenized DNA may form adouble-stranded mutagenized circular DNA intermediate, and digestingsaid DNA molecule for mutagenesis, wherein said digestion is mediated bya selection enzyme.
 2. The method according to claim 1, wherein saidselection enzyme digests methylated DNA strands and said selected DNAmolecule for mutagenesis is methylated.
 3. The method according to claim2, wherein said selected DNA molecule for mutagenesis is methylated invivo.
 4. The method according to claim 2, wherein said selected DNAmolecule for mutagenesis is methylated in vitro.
 5. The method accordingto claim 1, wherein the selection enzyme is a restriction endonuclease.6. The method according to claim 2, wherein the selection enzyme isDpnI.
 7. The method according to claim 1, wherein the linear cyclicamplification reaction is catalyzed by Pfu DNA polymerase.
 8. The methodaccording to claim 1, wherein the first and second mutagenic primers are5′ phosphorylated.
 9. The method according to claim 1, wherein thelinear cyclic amplification reaction is repeated for less than 20cycles.
 10. The method according to claim 1, wherein the first andsecond mutagenic primers are completely complementary to each other. 11.The method according to claim 1, said method further comprising thesteps, annealing said first mutagenized DNA strand and the secondmutagenized DNA strand so as to form a double-stranded mutagenizedcircular DNA intermediate, and transforming a host cell with saiddouble-stranded mutagenized circular DNA intermediate.
 12. A kit forintroducing a specific mutation into a selected DNA molecule formutagenesis, said kit comprising: a DNA polymerase, a selection enzyme,control first and second mutagenic primers, and control templates.
 13. Akit according to claim 13, said kit further comprising competent cells.14. A kit according to claim 14, said kit further comprisingconcentrated reaction buffers.
 15. A kit according to claim 13, whereinsaid DNA polymerase is Pfu DNA polymerase.
 16. A kit according to claim13, wherein said selection enzyme is a restriction endonuclease.
 17. Akit according to claim 17, wherein said restriction endonuclease is DpnI.